Infection with hepatitis B virus (HBV) is a major cause of liver disease worldwide and affects more than 1 million people in the United States. Hepatitis induced by hepatitis B virus infection is a complex and intricate process involving interactions of multiple host factors with the virus and/or the viral gene products. The HBV X (HBV) gene plays a crucial role in the life cycle and oncogenic potential of HBV. Since virus-host interactions are central to the pathogenesis of viral infection and host injury, this project aims to elucidate the cellular and molecular mechanisms of HBX-host interactions during HBV infection. We have previously shown that HBX interacts with the proteasome complex in vitro and in vivo. The 26S proteasome complex is the predominant cellular factor which degrades cellular proteins in both ubiquitin-dependent and -independent pathways. It has been implicated in the regulation of a variety of transcriptional and cell cycle factors, cellular stress response, and antigen presentation. HBX interacts with the proteasome subunits through a mutually competitive structural relationship. The crucial HBX sequences involved in the interaction with the proteasome complex are important for its function as a transcriptional coactivator. We have also shown that HBX functions as a substrate as well as an inhibitor of proteasome. Using the woodchuck model, we also demonstrated that the X-defective mutants of woodchuck hepatitis virus (WHV) are not completely replication defective, possibly behave like attenuated viruses in the woodchuck model. Animals inoculated with X mutants including those with no serologic evidence of infection were protected from the challenge, suggesting prior infection resulting protective immunity. To further study the role of the proteasome in the biology of HBX, we analyzed the effects of the proteasome inhibitors on the replication of wild-type and X-minus WHV and HBV using recombinant baculovirus or adenovirus expressing replicating HBV genome. We generated a recombinant baculovirus with CMV driven replication-competent WHV genome and another with the X-minus WHV genome by mutating the ATG start codon of the X gene to TTG. Recombinant adenoviruses expressing replicating HBV genome with and without a mutation inactivating the X gene were also used. In cells infected with either the recombinant baculovirus or adenovirus, the replication level of the X-minus construct was about 10% of that of the wild-type. When proteasome inhibitors were added, the replication of the wild-type virus was not affected, while the replication of the X-minus virus of either WHV or HBV was restored to the wild-type level. The inhibition of proteasome function by HBX may account for the multiple actions of HBX and may be an important feature of HBV infection, possibly in helping stabilize viral gene products and suppressing antigen presentation. We have also developed a novel yeast three-hybrid disruptor system to identify peptide aptamers that can block the interaction of HBX-proteasome as a novel antiviral approach. The effects of these peptide aptamers are being tested in the transactivation function of HBX and the replication of HBV in tissue culture.